Methods to improve immunogenicity of antigens and specificity of antibodies

ABSTRACT

A method of improving specific immune responses to smal immunogens, haptens, has been developed by changing the linkage between the hapten and carrier being used for immunization. High affinity antibodies to the hapten cotinine have been produced using this method. Antibodies to a glycated protein have also been developed, utilizing an immunogen which is composed of a glycated peptide mimic of the glycated peptide sequence which is the target epitope, wherein the peptide mimic is constructed to conformationally mimic the conformation of the peptide in the native protein, the peptide mimic contains no charged groups or other immunodominant group, and the peptide mimic is connected to a spacer sequence equivalent to a peptide spacer of between one and thirty amino acids in length, which serves to position the peptide epitope in a conformation that approximates its conformation in the native protein. In a further embodiment the peptide mimic and spacer are linked to a carrier molecule. This method has been used to produce an antibody to the glycated protein HbA 1 c, wherein the peptide mimic includes a valine modified by addition of a glucose molecule, an analog of Histidine which does not bear a charge in the immunizing structure, allows orientation of the peptide so that the immune response can be directed to the side of the peptide chain oriented oppositely to the ring, and is of a size that the conformation of the peptide mimics the conformation of the peptide in the native molecule, a leucine or an analog thereof which allows binding to an antibody preferentially recognizing Hb A 1 c such as 82D259, and a threonine or an analog thereof which allows binding to antibody number 82D259. In the example described below the histidine analog is 2-amino-3-flurylpropionyl, and the peptide is Fructosyl-Val-2-amino-3-furanylproprionic acid-Leu-Pro-Pro-Glu-Glu-Tyr-Tyr-Cys.

BACKGROUND OF THE INVENTION

[0001] This application claims priority to U.S. Serial No. 60/059,378entitled “Method to Improve Immunogenicity of Antigens and AntibodyResponses” filed Sep. 19, 1997 by Judith Fitzpatrick and to U.S. SerialNo. 60/090,458 entitled “Immunoassay and Antibody Specific forHemoglobin A1c” filed Jun. 24, 1998 by Judith Fitzpatrick and Regina B.Lenda.

[0002] Methods for making antibodies are well known and have becomeroutine for most antigens. However, some antigens, due to small size,conformation changes under different conditions, or lowimmunogenicity—for example, highly conserved protein or proteins whichare heavily glycosylated, have not been easy to make highly specificantibodies to.

[0003] A number of methods have been developed to address this problem.For example, it is well known that haptens or small molecules such aspeptides and drugs are not immunogenic unless conjugated to a protein.Such proteins are designated as carrier proteins and such conjugatedhaptens as immunogens. However, it has been discovered that conjugationcan alter not only the charge but also the conformation of the hapten,thereby generating antibodies that recognize the free hapten to a lesserextent than the immunogen.

[0004] The high immunogenicity of most linkers has also been a majorobstacle to generating monoclonal antibodies for haptens of small size,e.g. cotinine, for which both the yield of useful clones and theaffinities of available monoclonal antibodies are low. Thus, it hasproved very difficult to raise monoclonal antibodies to many drugs andto manufacture peptide vaccines that will induce neutralizing antibodiesto infectious agents.

[0005] Many commercial assays require highly specific antibodies,particularly for use in chromatographic assays where the result is to beindicative of a quantitative value, not just qualitative. For example,diabetes is a severe, life-threatening, chronic disease resulting froman impairment of the body's ability to turn glucose into usable energy.Type II diabetes is the most common form of diabetes. Up to 95 percentof the 16 million Americans with diabetes have Type II. It is also knownas adult-onset diabetes, as it usually develops in people over the ageof 45. In addition to age, weight and lack of physical activity orexercise, heredity also plays a role in a person's risk of having thedisease.

[0006] Heart disease, stroke, kidney disease, blindness, circulatory andnerve problems are linked to long-term, high levels of blood sugar(hyperglycemia). Co-morbid conditions often include hypertension, highcholesterol and triglycerides. Hemoglobin A1c (HbA1c) testing has greatimportance in the overall management of diabetes since HbA1c reflectsthe portion of glucose that attaches itself to hemoglobin. It has beenshown to accurately and reliably reflect long term levels (2-3 months)of chronic hyperglycemia. Therefore, while daily glucose monitoring isrequired for immediate intervention, HbA1c levels are considered a moreaccurate indicator of an individual's long term blood glucose levels. Inaddition to other in-office and at-home tests, the American DiabetesAssociation (ADA) recommends HbA1c testing four times a year forinsulin-treated patients and at least twice yearly for all otherpatients with diabetes, or as often as needed to help achieve goodglycemic control.

[0007] Just recently HbA1c has been approved for screening for diabetes.It is estimated that at some time during their lives approximately 10%of adults will develop adult onset diabetes. Most of these individualsare diagnosed after 10-15 years of hyperglycemia when the conditionresults in sugar in the urine. Damage is being done during theundiagnosed period. HbA1c screening could identify such individuals muchearlier. Research shows that the HbA1c test can provide information thatin many cases can help health care providers and patients developregimens that dramatically lower the risks for serious andlife-threatening diabetes complications, including blindness, kidneydisease and nerve damage. Each year, diabetes results in 54,000 leg andfoot amputations. Diabetes is the leading cause of end-state renaldisease (kidney failure). It is the fourth leading cause of death bydisease in the United States.

[0008] A landmark study known as the diabetes Control and ComplicationTrial (DCCT) revealed a direct correlation between high blood sugarlevels and the development of long-term complications in people withType I or insulin-dependent diabetes mellitus; there is no reason tobelieve that the effects of better control of blood glucose levels wouldnot also apply to patients with Type II diabetes. The DCCT also foundthat, through blood glucose and regular HbA1c testing, adjustments couldbe made in diet, exercise or insulin dosage to reducediabetes-associated risks. These include reductions in eye disease by up76 percent, kidney disease by 56 percent and nerve damage by 60 percent.

[0009] Due to the complexity of existing HbA1c tests, they are generallyperformed in clinical laboratories and at significant costs. Sincephysicians treating individuals with diabetes rely on this test for themanagement of the patient's disease, it is desirable for it to beperformed quarterly. Additionally, patients' interest in knowing theirHbA1c number has increased largely as a result of the DCCT study. MostType I (insulin dependent diabetic) know their HbA1c number just likethey know their blood pressure or cholesterol level.

[0010] Hemoglobin A1c (Hb A_(1c)) is one form of hemoglobin. It isidentical to Hemoglobin A_(o) (Hb A_(o)) with the exception that theN-terminal valine on the a chain is linked to C-1 of fructose throughthe amino group. This glycation causes a change in charge, whichresulted in its first identification as the A1c fraction on an ionexchange column procedure. The formation of valine-fructose residue isbelieved to result from the formation of a Schiff base between valineand glucose followed by an Amadori rearrangement. The process isirreversible and the ration of Ha A_(1c) constitutes 4-6% of the totalHb. In diabetes patients, the ratio increases two to three fold to6-15%.

[0011] The first step to develop an immunoassay in a Point of Care (POC)format to determine this ratio, i.e. Hb A_(1c)/total Hb, is to developan antibody that can discriminate between the native conformations ofHbAo and Hb A_(1c). Critical to providing a test for screening are lowcost reagents. The current assays for HbA1c entail expensive and/orcumbersome physical methods such as ion exchange and columnchromatography, or almost equally cumbersome and therefore non costeffective immunoassays. Thus while antibody based assays havetraditionally offered an economical alternative to physical methods thecurrent antibodies, as will be discussed below, currently availableantibodies do not offer the traditional advantages of specificity,economy and ease of use. Thus there is a need for an antibody that wouldoffer ease of use, economy and specificity. Such an antibody wouldenable both Point of Care testing and adaptation of a HbA1c assay to anyof many automated immunoassay systems.

[0012] There are several problems to be addressed when making anantibody to HbA1c or other antigens like HbA1c. The hemoglobin moleculeis a poor immunogen because the hemoglobin sequence is highly conservedand it is difficult to overcome tolerance of self. The peptide sequenceof the HbA1c epitope, hereafter the HbA1c epitope, is the same in mouseand human and most mammals: sheep have a different sequence in thisepitope region of the N terminal. It is difficult to overcome toleranceof self. Even though most animals do not form HbA1c, it is not possibleunder normal conditions to use Hb A_(1c) directly as the immunogen tomake an antibody that can discriminate HbAo from HbA1c since thedifference between A1c and Ao is only the addition of one glucosemolecule. Fructose has low immunogenicity and so the dominant immuneresponse is postulated to be to more immunodominant areas of theepitope.

[0013] A glycated site is not a good epitope: the HbA1c epitopecomprises less than 1% of the hemoglobin surface. Therefore one mustimmunize with a peptide. It is difficult to make an antibody to apeptide that has high affinity for the peptide sequence of the nativeprotein. The antibodies currently commercialized fall into twocategories, polyclonal and monoclonal.

[0014] Boehringer Mannheim (BM) markets a turbidometric assay kit whichutilizes a sheep polyclonal antibody. A sheep polyclonal prepared toHbA_(1c) whole molecule is described by Javid et. Al. (Brit. J.Haematology: 38:329-337 1978) and U.S. Pat. No. 5,646,255 to Klein, etal. The BM antisera was raised to the reported immunogen sequence“Fructose Val His Leu Thr . . . ” (Karl, et al. Klin. Lab 39:991-61993).It is probable that this antibody can be successfully raised in sheepbecause sheep do not have the same amino terminal sequence as othermammals and hence they are able to recognize as foreign andimmunologically response to the common mammalian N terminal sequence,“Val His Leu Thr”. The mouse in which monoclonals are raised has thesame sequence as humans and most other mammals and this probablyexplains why, when the same immunogen is used to immunize mice, that themajority, if not the only, antibodies that are produced, react with thedenatured form of hemoglobin (which is foreign) but not the nativeconformation (which is not foreign). It should also be pointed out thatall monoclonal antibodies are screened for in an Elisa format becausethat is the only truly economical method for performing all thescreening that must be done during the course of making a monoclonal.The Elisa plate is coated with the hemoglobin or protein: hemoglobin isnot in its native conformation, i.e., it denatures, when coated on anElisa plate. Thus even if the mouse did produce a few clones that hadthe potential to recognize the native conformation, the screeningprocess works to select for clones that recognize the denaturedconfiguration and thus against selection of a clone that would recognizenon denatured or native HbA1c.

[0015] The BM antibody does not show high specificity: In theexperimental Elisa system described herein, this sheep polyclonalantibody shows that about 10-20% cross reacts with HbA_(o) and reactsequally well with native (i.e. HbA_(1c) that is not specificallysubjected to denaturing conditions) and denatured HbA_(1c). The BMantibody is commercialized in a turbidometric assay. These are liquidbased assays that are run on an autoanalyzer and require less than 10minutes. In such a system one can often disregard low level crossreactivity because cross-reactive moieties having lower associationconstants exert less interference in shorter assays. However, it wouldbe difficult to utilize such an antibody in anything but an autoanalyzerassay, because to compensate for such high cross reactivity, all assayconditions, the time, pH, temperature, sample dilution, etc., must becarefully controlled, as they are on an autoanalyzer. In a point of careassay, it is not possible to dilute the sample or to add large amountsof buffering materials or to carefully control time and temperature.Thus cross reactivity becomes a much larger problem under assayconditions that are not ideal.

[0016] A further disadvantage of the BM polyclonal antibody is that itis not cost effective. The kit provides the exceptionally large amount,40 μg, of purified antibody for each assay. Polyclonal antibody thatmust be purified and provided at such high concentration is veryexpensive. Point of Care assays, which usually require much largeramounts of antibody than laboratory assays, generally have less than 1μg/assay. The large amount of antibody required by the BM test isprobably due to low affinity of the antibody. Low affinity antibodygenerally means that the assay lacks a high degree of specificity.Indeed, it is well documented that all the currently commercializedimmunoassays for HbA1c are lacking in specificity; i.e. they do notdiscriminate as well as required between various modifications of the Nterminal valine.

[0017] Thus, while polyclonal antibody raised in sheep has the advantagethat it can recognize native HbA1c sequence, it has the disadvantagesthat it is very expensive and that it lacks specificity and thus islimited to formats that can compensate for these restrictions on itsperformance. More importantly, polyclonal antibodies to one discreteconformational epitope cannot provide the consistency that is requiredby today's clinical laboratory standards, i.e. by definition, polyclonalantibodies contain many clones of different affinity and each animal ateach bleed provides a mix of clones that unique to that bleed. Whenthere is only one epitope and that epitope is conformational, polyclonalantibodies generally provide unacceptable variations in reagents fromlot to lot.

[0018] A monoclonal antibody to HbA1c would have the potential toovercome the cost and consistency problem of polyclonal antiserum butthus far all available and described monoclonals recognize altered HbA1cand require either a 10 minute denaturation process or proteasetreatment to render the sample suitable for testing with the antibody.The requirement for pretreatment of sample precludes the adaptation ofthese antibodies to Point of Care Tests; non-laboratory personnel in anon-laboratory environment cannot be expected to treat the sample.Pretreatment also greatly limits the usefulness of such antibodies inscreening assays and pretreatment adds significant cost and complexityto the test.

[0019] Monoclonal antibodies specific for the glucosylated N-terminalpeptide residue in HbA_(1c) are described in U.S. Pat. No. 4,727,036 toKnowles. These antibodies are hereafter referred to as the MilesAntibody. The Miles antibody was produced using the glycated N-terminalfragment of â chain as the immunogen. The first 15 amino acids of achain N-terminal areVal-His-Leu-Thr-Pro-Glu-Glu-Lys-Ser-Ala-Val-Thr-Ala-Leu-Trp. The MilesAntibody was produced to a peptide sequence that included the first 8amino acids: BM also utilized only the first 7 or 8 amino acids. Miles'HbA_(1c) specific antibody is a monoclonal antibody which reacts onlywith denatured HbA_(1c) (the kit requires 4 minutes with a chaotropicagent and it interacts with HbA1c immobilized on microtiter plate. TheMiles antibody discriminates with great specificity between HbA_(o) andHbA1c when these proteins are denatured. This antibody shows almost nocross reactivity with HbA_(o) in our experimental Elisa assay. Howeverit does not function as a diagnostic reagent unless the reagents aredenatured. The patented immunogen for the Miles monoclonal isfructose-Val-His-Leu-Thr-Pro-Glu-Glu-Lys-Tyr-Tyr-Cys. (Tyr-Tyr iscommonly used to obtain spacing of immunogen from the protein carrier).This is essentially the same immunogen described in the literature forproducing polyclonal antibody to HbA1c and used by BM to produce sheeppolyclonal. This monoclonal antibody recognizes the amino terminus indenatured HbA1c. Thus the sample requires pretreatment to allowinteraction with the antibody. This pretreatment step renders the testof limited economic value. Further the literature indicates that theantibody recognizes other modifications of the terminal valinecontaining peptide: this indicates that the antibody recognizesmodification of the N terminal, not the specific modification.

[0020] Generally speaking, antibodies made to linear peptide analogs ofepitopes are of low affinity and thus lack specificity i.e. they willshow high cross reactivity. In this case that means that the antibodymade to HbA1c would be expected to recognize both HbAo and HbA1c. Themonoclonal antibody described in patent U.S. Pat. No. 4,647,654 toKnowles, marketed by Miles, does not distinguish between Ao and A1cunless they are denatured; in the denatured form the peptide appears tothe antibody as similar to the immunogen.

[0021] It is therefore a first object of the present invention toprovide a method for making antibodies to immunogens that have lowimmunogenicity.

[0022] It is another object of the present invention to provide methodsand reagents to enable generation of high titer antibodies to preferredepitope conformations, especially those where the conformation isaltered by conjugation to carrier or by denaturation.

[0023] It is a second object of the present invention to produce amonoclonal antibody that reacts with antigens such as native HbA1c, thatis both more accurate and sensitive than the antibodies used incurrently available tests, and yet at the same time is cheaper toproduce and use.

[0024] It is a further object of the present invention to provide anantibody that is useful in a point of care test and thus does notrequire any treatment and must react with the native molecule.

[0025] It is still another object of the present invention to provide amethod and reagents to quickly and inexpensively measure antigens suchas Hb A1c and to determine the ratio of antigens such as Hb A1c and HbAo.

SUMMARY OF THE INVENTION

[0026] Methods are described herein to enhance the specificity ofmonoclonal antibodies to antigens characterized by low immunogenicity orwhich do not elicit production of highly specific antibodies with littlecross-reactivity. Examples of such antigens include glycosylatedproteins, proteins which are highly conserved among species, and verylow molecular weight proteins which are immunogenic only as haptensconjugated to carrier molecules.

[0027] In a first method, the initial immunization is performed with afirst immunogen and the second, “boosting” immunization is performedwith a slightly different immunogen which shares in common with thefirst immunogen the epitope(s) to which an antibody response is desired.In a second method, the immunogen is modified so that immunodominantepitopes are altered, resulting in an antibody response to an epitopewhich is present in both the denatured or native proteins or which isobscurred in the more immunogenic derivative used for the initialimmunization.

[0028] In the examples using cotinine and hemoglobin, immunizationprotocols are described in which the initial immunization is performedwith one immunogen and boosting is done with a second immunogen of adifferent structure. In the first embodiment, the structural alterationis confined to the linker while the hapten and the carrier proteinremain unchanged. The method thus overcomes problems resulting fromconformational changes, linear-specific antibodies and lowimmunogenicity of haptens. This protocol was found to produce superiorantibody responses to, and be particularly useful and effective, withsmall haptens such as cotinine.

[0029] A method of producing an antibody to a glycated protein has alsobeen developed, which utilizes an immunogen which is composed of aglycated peptide mimic of the glycated peptide sequence which is thetarget epitope within a larger protein, wherein the peptide mimic isconstructed to conformationally mimic the conformation of the peptide inthe native protein, the peptide mimic contains no charged groups orother immunodominant group, and the peptide mimic is connected to aspacer sequence equivalent to a peptide spacer of between one and thirtyamino acids in length, which serves to position the peptide epitope in aconformation that approximates its conformation in the native protein.In a further embodiment the peptide mimic and spacer are linked to acarrier molecule. This method has been used to produce an antibody tothe glycated protein HbA1c, wherein the peptide mimic includes a valinemodified by addition of a glucose molecule, an analog of Histidine whichdoes not bear a charge in the immunizing structure, allowing orientationof the peptide so that the immune response can be directed to the sideof the peptide chain oriented oppositely to the ring, and is of a sizethat the conformation of the peptide mimics the conformation of thepeptide in the native molecule, a leucine or an analog thereof whichallows binding to an antibody preferentially recognizing Hb A1c such as82D259, and a threonine or an analog thereof which allows binding toantibody number 82D259. In the example described below the histidineanalog is 2-amino-3-flurylpropionyl, and the peptide isFructosyl-Val-2-amino-3-furanylproprionicacid-Leu-Pro-Pro-Glu-Glu-Tyr-Tyr-Cys.

[0030] In a preferred method of immunizing to a glycated peptide linkedto a carrier protein, the portion of the peptide that serves to link thepeptide to the carrier protein is selected to provide minimal antigeniccompetition for immune response and to maintain the epitope portion ofthe molecule in the configuration that it appears on the surface of themolecule. Further in the method of immunizing to a glycated peptidelinked to a carrier protein, the method of linkage of the peptide to thecarrier protein is changed from the first to the second immunizing dosesto avoid boosting to the linker specific antibodies and to avoidboosting to a linker induced epitope conformation.

BRIEF DESCRIPTION OF THE DRAWINGS

[0031] FIGS. 1A-1D are structures of cotinine derivatives: cotininecaproic acid (FIG. 1A), CPE cotinine (FIG. 1B), hydroxymethyl contininehemisuccinate (HCH) (FIG. 1C), and carboxycotinine (FIG. 1D).

[0032]FIG. 2 is a graph of the antibody titer produced in mice immunizedwith carboxy-cotinine-KLH.

[0033]FIG. 3 is a graph of antibody binding to cotinine (diamonds) orcarboxy-cotinine (squares) at concentrations of between 0 and 8micrograms/ml.

[0034]FIG. 4 is a graph of the interaction of two monoclonal antibodies,Mab 57F126 and 57F133 with cotinine (one microgram/ml, open squares) andurine samples (positive—containing cotinine, closed squares;negative—not containing cotinine, lines at baseline).

[0035]FIG. 5 is a graph of the antibody titer produced by immunizationwith a first immunogen followed by a boost with a second immunogen whichdiffered from the first immunogen only by the structure of the linker.The first immunogen had hdyroxymethyl heme succinate linking cotinine tokeyhole limpet hemocyanine (KLH). The second immunogen included acarboxyl group linking continine to KLH. The first boost was three weeksfollowing initial immunization. Ten micrograms/mouse was used for eachimmunization.

[0036]FIG. 6 is a graph of antibody binding of several monoclonalantibodies with cotinine (one microgram/ml, open squares) and urinesamples (positive—containing cotinine, closed squares; negative—notcontaining cotinine, lines at baseline).

[0037]FIG. 7A is Hyperchem model of the native sequence of the glycatedVal-His-Leu-Thr-Pro-Glu-Glu, the histidine residue is prominent.

[0038]FIG. 7B is a Hyperchem model showing that when the histidineresidue is replaced by phenylalanine, the residue is still prominent andnot in the interior position one would imagine if the charge wereneutralized by ionic interaction with an interior residue.

[0039]FIG. 8A is a Hyperchem model showing positioning the His imidazolering in the interior of the folded peptide, by replacing the threonineat residue four with a proline to position the imidazole ring ofhistidine in an interior position while prominently displaying theglycated valine and backbone of histidine.

[0040]FIG. 8B is a Hyperchem model which, while not being a model of the2-amino-3-flurylpropionyl (Her) but utilizing phenylalanine as a Hermimic, indicating that one will achieve the proper orientation andsteric conformation since the phenyalanine ring is bulkier than a furanring and one would therefore expect that with the furan ring the ringwould assume a more closed position similar to FIG. 8A.

[0041]FIG. 9 is a graph showing screening of hybridomas from fusion 82Dfor clones reactive with HbA1c (but only minimally reactive with HbAo)in an Elisa System. Plates were coated with purified HbA1c or, HbAo. A2.5 fold dilution of fusion supernatant was incubated for 1 hourfollowed by wash and 1 hour incubation with Peroxidase labeledanti-mouse. High absorbance value correlates with high recognition ofthe plate coating material.

DETAILED DESCRIPTION OF THE INVENTION

[0042] Antigens

[0043] The following definitions are provided:

[0044] A hapten is a molecular moiety of less than 10,000 molecularweight, most often of less than 2,000 molecular weight.

[0045] A hapten analog is used herein to refer to a hapten which hasbeen modified but retains essentially the same immunologicalcharacteristics of the hapten of interest.

[0046] An epitope is a chemical conformation, for example 4 to 12 aminoacids, recognized by an antibody.

[0047] A carrier molecule is a large molecule, generally greater than40,000 molecular, for example, a large peptide, protein or particle,which is sufficiently large that multiple haptens can be conjugated toit. Examples include polylysine, keyhole limpet hemocyanine, and bovineserum albumin.

[0048] An antigen elicits an immune response in the form of an antibody.

[0049] A linker is a molecule connecting two other molecules, forexample, the antigen and a carrier molecule.

[0050] A linkage is the direct chemical coupling between two molecules,for example, a hapten and a carrier molecule, in which the conformationof the hapten may be influenced by the nature of the chemical linkage.

[0051] Antigens can be proteins, synthetic organic molecules, metals,and sugars, alone or in combination with proteins. Preferred antigensfor use in the methods described herein glycated hemoglobin and otherglycated proteins, amphetamines and other drugs which do not elicithighly specific antibodies or which might be poorly immunogenic. Anotherpreferred antigen is where the desired response is to one or moreepitopes in a protein, for example, in a vaccine, not to all of theepitopes present in the protein.

[0052] Methods of Modifying Antigens to Enhance Immunogenicity

[0053] Conjugation

[0054] Numerous conjugation methods are known in the art and aredescribed, for example, by G. T. Hermanson in “Bioconjugate Techniques”,Academic Press, 1996. Briefly, conjugations of a hapten to a carrier isgenerally effected by means of linkers or, more appropriatelycross-linkers, which consist of linear molecules of various lengthbearing reactive functional groups at both ends. In homobifunctionallinkers (i.e. glutaraldehyde) the two functional groups are identical:in heterobifunctional linkers, they are different. The detailedconjugation chemistries are well known. The final conjugation productcan be thus depicted as hapten_(x) --linker_(y) --carrier_(z) (H-L-C),for example, where H is a cotinine derivative, L is a crosslinker and Cis keyhole limpet hemacyanin (KLH). The dashes represent covalent bonds;x and y are identical and generally much larger than z.

[0055] Derivatization

[0056] Immunogens can be modifed as described below by substitution ordeletion of specific amino acid residues, chemical coupling of blockingagents, sugars, linkers and/or carrier proteins, and other methods knownto those skilled in the art. These are screened for binding toantibodies which are known to have a desired specificity and the resultscompared with binding to the molecule which is to be quantitated, forexample, using standard immunoassays or other means of quantitation asdemonstrated in the example.

[0057] Methods for Producing Antibodies

[0058] The science, or more properly, the art of antibody production hasprogressed over the past decades. Well established and tested proceduresare provided, e.g. in “Antibodies-A Laboratory Manual”, E. Harlow and D.Lane, Cold Spring Harbor, 1988 and in “Monoclonal Antibodies”, R. H.Kennett et al., eds., Plenum Press, 1980. The more theoretical aspectsof antibody production are discussed in “Antibody Affinity”:Thermodynamic Aspects and Biological Significance”, M. W. Steward and J.Steensgaard, CRC Press, 1983, the teachings of which are incorporatedherein by reference.

[0059] The present invention will be further understood by reference tothe following non-limiting examples making antibodies to cotinine andhemoglobin.

EXAMPLE 1 Preparation of Cotinine Derivatives Which Yield More SpecificAntibodies to Native Cotinine

[0060] Over the past 15 years, Serex has successfully produced severalpolyclonal antibodies for cotinine, but has consistently failed innumerous attempts to raise monoclonal antibodies using conventionalprotocols. Other investigators, e.g. J. J. Langone, J. Immunol. Meth.90, 203-213 (1986), also obtained cotinine monoclonal antibodies, butthese antibodies are linker specific. U.S. Pat. No. 5,164,504, assignedto Abbott, disclosed preparation and use of two cotinine immunogenswhich yielded antibodies with very low titers. Monoclonal cotinineantibodies are commercially available, but have little or no practicalutility.

[0061] Possible explanations for the problems with cotinine are itssmall size and low immunogenicity. Cotinine, a major metabolite ofnicotine, is also structurally related to several nicotinic acidderivatives which are present in mammalian plasma and tissues. Usingconventional immunization protocols, Serex has never succeeded inproducing monoclonal or polyclonal antibodies in mice or rabbits withtiters of >1:5000 even after several boosts over a period of six months.Furthermore, the resultant antibodies consistently reacted with thelinker and generally were of low affinity. The monoclonal antibodydescribed by Langone (cited above) reacted strongly with the linkerportion as evidenced by high crossreactivity with the drug metyrapone.

[0062] In contrast, the approach described above yielded concentrationsof monoclonal antibodies equivalent to titers of 1:100,000 after asingle boost with the second immunogen which differed from the firstimmunogen only in the structure of the linker (L₂ versus L₁ in H-L-C).This procedure yielded many clones of high titer and specificity. Thedramatic improvements in immune responses obtained with the protocolwere totally unexpected and are contrary to conventional wisdom whichprescribes boosting with the same type and preferably the identicalpreparation or lot of immunogen. It is believed that this methodovercomes the problem of linker specific antibody as well as linkerspecific conformation of hapten and propose that it will prove a usefulstrategy for immunization with any small molecule drug or peptide whichis conjugated to a carrier for immunization.

[0063] It is believed that boosting with the altered immunogen of thisimmunization protocol stimulates clones specific for H and C, which arecommon to both immunogens, but not clones specific for L₁ which remainat the low pre-boost levels. Boosting with the L₂ linker in the secondimmunogen mounts a weakened response to L₂, in relation to the L₁response, whereas there is the expected dramatic increase in theresponses to H (and C), thus yielding antibody clones with high titersand recognition for cotinine, but very low recognition of both linkers.

[0064] Preparation of Cotinine Derivatives

Preparation of Cotinine Caproic Acid

[0065] As shown in FIG. 1A, the mixture of cotinine (106 mg) andbromohexanoic acid (117 mg) in 1 mL of DMF was heated at 100° C.overnight. The mixture was allowed to cool to room temperature and thesolvent was removed under reduced pressure. The residue was rinsed withCH₂Cl₂ three times and the produce (110 mg) was obtained as an off whitefoam.

Preparation of Carboxyphenylethyl Cotinine Bromide (CPE Cotinine)

[0066] As shown in FIG. 1B, a solution of cotinine (95 mg) andbromoethyl benzoic acid (123.4 mg) in DMF (1 mL) was heated at 100° Covernight. The solvent was removed and the residue was washed withCH₂Cl₂ three times to remove unreacted starting materials. The residue(154 mg) was used without further purification.

Preparation of Carboxycotinine Methyl Ester

[0067] To a suspension of carboxycotinine (344 mg) in 10 mL of MeOH wasadded 0.5 mL of concentrated H₂SO₄. The resulting solution was stirredovernight at room temperature. Solid NaHCO₃ was added to neutralize thesolution and the solution was filtered. Methanol was removed. Theresidue was redissolved in CH₂Cl₂, washed with saturated NaHCO₃, driedover Na₂SO₄. Solvent was removed and the white powder (270 mg) was usedwithout further purification.

Preparation of Hydroxymethyl Cotinine

[0068] To a solution of carboxycotinine methyl ester (270 mg) in MeOH(10 mL) was added 400 mg NaBH₄ in portions. The suspension was stirredat room temperature for 4 hr. 1 mL of 20% HCl was added and was stirredfor 15 min. The pH was adjusted to 10 with NaHCO₃ powder and NaOHsolution. MeOH was removed and the aqueous was extracted five times withCH₂Cl₂. The organic layer was dried over Na2SO₄ and the solvents wasremoved. The residue was purified by flash chromatography (10% MeOH inCH₂Cl₂) to give 188 mg product as an oil.

Preparation of Hydroxymethyl Cotinine Hemisussicinate (HCH)

[0069] As shown in FIG. 1C, to a solution of hydroxymethyl cotinine (33mg) in 3 mL of benzene was added succinic anhydride (16 mg) and thesolution was heated at 70° C. overnight. A white precipitate formed.Cooled to room temperature and decanted the benzene. The white powderwas washed with ether a few times and the remaining white powder (43 mg)was used without further purification. TLC and NMR data confirmed theassigned structures. Purities of the cotinine derivatives are >90% byTLC.

[0070] Preparation of Cotinine Immunogens

Preparation of Carboxy-Cotinine-KLH Conjugate

[0071] A solution of carboxy-cotinine (22 mg), N-hydroxy-succinimide,NHS (11 mg) and N,N′-dicyclohexylcarbodiimide, DCC (22 mg) indimethylformamide, DMF (1 ml) was stirred in room temperature for 1hour. The mixture was then added to a solution of Keyhole LimpetsHemocyanin, KLH (20 mg) in 0.1 M Carbonate Buffer, pH 9 and incubatedfor four hours, then dialyzed against four changes of PBS.Carboxy-Cotinine is depicted in FIG. 1D.

Preparation of Cotinine-Caproic-KLH Conjugate

[0072] A solution of cotinine-caproic acid, CCA (3 mg), NHS (6 mg) andDCC (12 mg) in DMF (0.6 ml) was stirred at room temperature for 1 hour.The mixture was then added to a solution of KLH (5 mg) in PBS (3 ml).The mixture was stirred for four hours at room temperature and thendialyzed against four changes of PBS.

Preparation of Carboxyphenylethlyl-Cotinine-KLH Conjugate

[0073] A solution of carboxyphenylethyl-cotinine, CPEC (2 mg), NHS (11.2mg) and EDC (2.4 mg) in DMF (0.1 mg) was stirred at room temperature for1.5 hour. The mixture was then added to a solution of KLH (10 mg) in0.083M Phosphate Buffer in 0.9M NaCl, pH 7.2 (1 ml). After overnightincubation, the mixture was dialyzed against four changes of PBS.

Preparation of Hydroxymethyl-Cotinine-Hemisuccinate-KLH Conjugate

[0074] A solution of Hydroxymethyl-Cotinine-Hemisuccinate, HCH (3.2 mg),NHS (3.2 mg) and DCC (6 mg) in DMF (2.3 ml) was stirred at roomtemperature for 1 hour. The mixture was then added to a solution of KLH(10 mg) in 0.083 M Phosphate Buffer in 0.9M NaCl, pH 7.2 (1 ml). Afterovernight incubation, the mixture was dialyzed against four changes ofPBS.

[0075] Immunization Protocol

[0076] Mice, Balb C or Swiss Webster, were injected first with cotininederivative-KLH conjugate at 10 microgram per mouse with CFA. After threeweeks the mice were boosted with 10 microgram of immunogen per animalwith IFA. Two weeks after the first boost, the mice were bled and theantisera were tested in Elisa for Anti-cotinine antibody titer. Secondand subsequent boosts, if used, were done at three week intervals with10 microgram of immunogen per dose. Testing was done two weeks aftereach boost.

[0077] Four different immunization routes were used:

[0078] Route A: First injection and all three boosts were done with thesame immunogen, Carboxy-cotinine-KLH.

[0079] Route B: First injection and next three boosts were done with thesame immunogen, Cotinine-caprioc-KHL. The fourth boost was done withimmunogen CPEC-KLH.

[0080] Route C: First injection was done with Carboxy-cotinine-KLH andfirst boost was done with HMCH-KLH.

[0081] Route D: First injection was done with HMCH-KLH and first boostwas done with Carboxy-cotinine-KLH.

[0082] Immunization Route A

[0083] Three Balb C mice immunized. Antisera were tested for antibodytiter in microtiter plate Elisa using carboxy-cotinine derivativeconjugated to bovine gammaglobulin as a solid phase. The amount ofantibody bound to the plate was detected by goat anti-mouse IgG antibodylabeled with peroxidase. Peroxidase was assayed with TMB substrate.Results on antisera titration are presented in Table 1 and FIG. 2.Subsequent boosts did not improve titers. After three boosts, the mousewith the highest antibody titer was selected for fusion. Fusion yieldeda cell line 6F4.3.1 which produced monoclonal antibody to cotinineimmunogen. The results are shown in Table 2 and FIG. 3.

[0084] Monoclonal antibody obtained with immunization route where thefirst injection and all boosts were done with the same immunogen,Carboxy-Cotinine-KLH, showed no specificity to free cotinine. Theinteraction of the antibody with Carboxy-Continine may be due tospecificity for the carbonyl linker.

[0085] Immunization Route B

[0086] Five Babl C and five Swiss Webster mice were immunized. Aftereach boost the anitsera were tested for antibody titers and forspecificity to continue. Antisera tested on CCA-BSA plates showedincreasing antibody titers that reached level of 1:400,000 after thethird boost. Testing for specificity was done in Elisa by determinationof antibody binding to plates in the presence of free cotinine ornegative and positive smokers urines. In this assay the plates werecoated with Carboxy-continine derivative-BGG conjugate. After the thirdboost with the same immunogen CCA-KLH, the antibody showed nospecificity to cotinine but interaction with positive urine. After thefourth boost with CPEC-KLH testing showed a great improvement inspecificity to cotinine and stronger interaction with positive urine.Results are presented in Table 3. TABLE 3 Evaluation of mouse serumafter a boost with changed immunogen. Serum at 1:100 Serum at 1:200dilution dilution after Third Boost after Fourth Boost Sample A 450 nm %BO A 450 nm % BO Negative Urine 0.465 100 1.384 100 Positive Urine 0.29664 0.703 51 Continine, 1 0.572 123 0.947 68 ug/ml

[0087] The fourth boost increased antibody titers and improvedspecificity to Cotinine.

[0088] Two cells lines (57F126 and 57F133) producing monoclonal antibodywere obtained in this study. The antibodies interacted sgtrongly withsmokers urines and showed also some reactivity with free cotinine asshown by FIG. 4.

[0089] Immunization Route C

[0090] Five Balb C mice were immunized in this study. After the firstboost, the antisera were tested in Elisa on plates coated withCarboxy-cotinine-BGG conjugate. The antibody titers were very low, only1:100. One mouse showed no response.

[0091] Immunization Route D

[0092] Five Babl C mice were immunied in this study. After the firstboost, the antisera were tested in Elisa on plates coated with HMCH-BGGconjugate. The antibody titers were very high above between 1: 100,000and 1 :100,000 (see FIG. 5).

[0093] Testing for specificity was done in Elisa by determination ofinhibition if antibody binding to plates in the presence of freecotinine and negative and positive urine samples. In this assay plateswere coated with CPEC-BGG conjugate. Antiserum from mouse # 60.2 had thebest characteristics and was selected for fusion. The antiserum showedtotal inhibition of antibody binding to the plate by positive smokersurine and 90% inhibition by free cotinine (see Table 4). TABLE 4 Testingof mouse serum in Elisa. Serum at 1:100 dilution after Third BoostSample A 450 nm % BO Negative Urine 0.349 100 Positive Urine 0 0Continine, 1 ug/ml 0.033 9

[0094] Fusion yielded 17 cells which produced cotinine specificmonoclonal antibody. Ascites were produced from the six best cellslines; 57G5, 57G9, 57G11, 57G15, 57G16 and 57G17. The results are shownin Table 5 and FIG. 6. TABLE 5 Testing of hybridoma supermatants at 1:20dilution in Elisa with free cotinine and urine samples. Absorbance at450 nm Cotinine Sup # Neg. Urine Post. Urine (1 μg/ml) 57G5  1.578 0.1450.151 57G9  0.775 0.11 0.079 57G11 0.379 0.101 0.093 57G15 0.405 0.090.073 57G16 1.054 0.128 0.108 57G17 0.205 0.1 0.072

EXAMPLE 2 Preparation of Hb A1C Immunogens and Immunization Protocols toProduce Highly Specific Antibodies

[0095] It is difficult to get an antibody to distinguish between HbAoand HbA1c, as the difference is only one small sugar molecule. Sugarsare poor immunogens and indeed the antibody methods on the marketgenerally do not distinguish between a sugar and any other small changeat the sugar site. General methods have therefore been developed toincrease the immunogenicity of these glycoproteins and other poorimmunogens, by altering the immunogenicity of the immunodominantepitope(s). The method utilizes an immunogen which is composed of aglycated peptide mimic of the glycated peptide sequence which is thetarget epitope, wherein the peptide mimic is constructed toconformationally mimic the conformation of the peptide in the nativeprotein, the peptide mimic contains no charged groups or otherimmunodominant group, and the peptide mimic is connected to a spacersequence equivalent to a peptide spacer of between one and thirty aminoacids in length, which serves to position the peptide epitope in aconformation that approximates its conformation in the native protein.The peptide mimic and spacer can be linked to a carrier molecule. Thismethod has been used to produce an antibody to the glycated proteinHbA1c, wherein the peptide mimic includes a valine modified by additionof a glucose molecule, an analog of Histidine which does not bear acharge in the immunizing structure, allows orientation of the peptide sothat the immune response can be directed to the side of the peptidechain oriented oppositely to the ring, and is of a size that theconformation of the peptide mimics the conformation of the peptide inthe native molecule (2-amino-3-flurylpropionyl), a leucine or an analogthereof which allows binding to an antibody preferentially recognizingHb A1c such as 82D259, and a threonine or an analog thereof which allowsbinding to antibody number 82D259.

[0096] One of the problems with all the antisera based tests on themarket is that they do not discriminate well between the variousmodifications that occur at the N terminal valine e.g. they do notdiscriminate well between an acetylated valine and one which isglycated. This is probably due to the fact that the antibody recognizeslinear sequence and the amino acids are immunodominant to the sugar. TheBM antibodies generated to a Hb A_(1c) immunogen show highcross-reactivity with Hb A_(o). Solid phase and solution phase bindingstudies were conducted to clarify how this antibody distinguishedbetween the denatured and the native protein. In solid phase studies,the HbA1c protein was coated on a microtiter plate where the hydrophobiccharacter of the plastic causes the denaturation of the protein,therefore exposing the linear peptide recognition domain. For solutionphase studies, the protein was captured by an antibody on the platesduring the assay, so that no denaturation occurred. In both cases, theantibody interacted with the anti Hb A_(1c) antigen with no dramaticdifference. There were two possibilities: 1. The purified Hb A_(1c) wasdenatured during purification. 2. The antibody recognizes no differencebetween the denatured and native form of the protein 3. Exposure to thedetergent used in lysing the red blood cells and during the Elisa essaywas sufficient to denature the liquid phase HbA1c.

[0097] It was predicted that the HbA1c epitope was present on thesurface of the native protein but that in the denatured state theepitope appeared quite different and contained an immunodominant moietythat was not present in the native conformation of HbA1c and HbAo. Itwas reasoned that as long as this immunodominant moiety was present theantibodies generated would not recognize a sugar and hence would beunable to distinguish between HbA0 and HbA1c in the native form.

[0098] A strategy was developed to create unique immunogens that resultin a monoclonal that recognized native HbA1c. Since HbA1c isdistinguishable from HbAo on a column it seems reasonable to assume thatthe epitope is accessible in the native molecule. Molecular models showthe epitope on the surface and the BM antibody recognizes the epitope inat least nearly native format. It was reasoned that the conformation ofthe peptide was different in the native as opposed to the denaturedpeptide and that this might be result of the histidine: as a rule,charged groups are more immunogenic than non-charged groups.

[0099] An alternative explanation is that the Miles antibody recognizesthe charged His side and this is the inside non-accessible face of thepeptide in the native protein, whereas the BM antibody recognizes theoutside or non charged side of the peptide. An immunogen was designed torecognize that one hypothesize to the configuration of the peptide inthe native protein.

[0100] It was reasoned that it was needed to defocus the immune responsefrom the linear peptide and the histidine and to focus the immuneresponse on the fructose modification of the N terminal valine and thenative conformation of the peptide. If the difference between the nativeHb and denatured Hb molecule was the histidine charge, that charge couldaffect orientation (which was the result of histidine having a saltbridge type of interaction with an internal portion of the molecule) ofthe histidine. Thus it was reasoned that in the native form of Hb theconformation of the peptide was such that the imidazole ring orientedthe histidine so that it was “tacked” by its charged ring nitrogen tothe body of the protein. Thus the ring “faced the protein interior”. Toassure that this orientation and space filling conformation was achievedand that the charge did not become immunodominant an analog that had nocharge was synthesized. Phenylalanine was also promising but was notused since an analog with the closest “space-filling” dimensions wasneed to get the highest specificity with a weak immunogen. It wasreasoned that this non-charged peptide would interact with an antibodywithout the binding strength conferred by charge and thus one couldimprove immunogenicity by improving the “fit” or exactly mimicking thespatial relationships that would exist between the epitope and thebinding site and thus it was aimed to provide an analog with the closest“space filling” dimensions reasoning that this would result in aconformation that was closest to that of the epitope on the surface ofthe molecule.

[0101] It was hypothesized that the Histidine residue might beimmunodominant in the peptide immunogen sequence, but that in the nativeprotein in the Histidine could be in a salt bond with another residue.Hence an antibody that depended on interaction of a negatively chargedbinding site residue with the positively charged His might not recognizenative HbA_(1c). This hypothesis was tested utilizing two peptides incompetitive assays:

[0102] Val-His-Leu-Thr-Pro-Glu-Glu . . . termed (His) and

[0103] Val-Phe-Leu-Thr-Pro-Glu-Glu . . . termed (Phe).

[0104] It was reasoned that Phe filled a similar space as His but had nocharge and so could be used to monitor the dependence of antibodyaffinity on the presence of a charged His residue. In this experiment,HbA_(1c), anti HbA_(1c) BM and Miles antibodies, and the peptides wereincubated in microtiter plate wells previously coated with rabbitanti-Human Hb. Both peptides are shown to equally inhibit theinteraction of BM antibody with HbA_(1c) as well as HbA_(o). However,when the Miles antibody was tested, there was a big difference ininhibition of the interaction of Mab with HbA_(1c), the His-peptideshowed strong inhibition while the Phe-peptide did not interfere. Thedata suggests that His charge is integral to the Miles' antibody bindingto HbA_(1c), however, it is not important in BM antibody's interactionwith HbA_(1c). This suggests that the Miles antibody selectivity for thedenatured form of HbA_(1c) is actually a requirement for a chargedaccessible His and the BM antibody which binds equally well to bothdenatured and native HbA_(1c) is not affected by the charge on His. Thissuggests that one should not use a peptide with a charged His residue.The molecular models discussed in detail below and shown in FIGS. 7A and7B and 8A and 8B support these conclusions.

[0105] This immunogen is a peptide-protein conjugate containing thesequence Fructose Val H Y Y . . . -R. Where H is an uncharged analog ofHistidine and Y is the native or other peptides providing up to 20 ormore amino acids and R is a protein. In a preferred embodiment, H is astructural analog of His with imidazole ring substituted by a furanring, 2-amino-3-furylproprionic acid, hereafter, “Her”.

[0106] It has been shown that the peptide with H=Phe interacts with BMantibody similar to the peptide with His. The advantage of using Her inthe immunogen is to eliminate the charge on the second residue,therefore focusing the immune recognition on fructose-Val-uncharged HisLeu Thr while maintaining the size of the imidazole ring. It wasbelieved that this approach had the potential to generate an antibody torecognize native HbA_(1c).

[0107] It was hypothesized that the charge on the histidine in thelinear peptide was the immunodominant epitope when one immunized withthe linear peptide and that in the native molecule this charge could beneutralized by interaction with another amino acid. In this model thecharge carrying nitrogen of the imidazole ring is oriented towards theinterior of the molecule. Therefore a peptide with an analog ofhistidine with neutral charge (hereafter “Her”) was synthesized. Thepeptide was modeled using a molecular modeling program to provide apeptide that oriented the charge carrying position towards the interiorof the peptide.

[0108] The immunogen which was designed had the sequence:Fructose-Val-2-amino-3-flurylpropionyl-leu-Pro-Pro-Glu-Glu-Tyr-Tyr-Cys

[0109] 1 2 3 4 4 5 6 6 7 8

[0110] which can form part of a longer immunogen having the followingformula:

Fructose-Val-Her-3-4-5-6 . . . -X-R,

[0111] where residue 1 is a valine (or analog thereof) modified byaddition of a glucose molecule;

[0112] where residue two is an analog of Histidine that does not bear acharge in the immunizing structure;

[0113] where residues three to twenty allow the orientation of thepeptide so that the immune response can be directed to the side of thepeptide chain oriented oppositely to the ring; and

[0114] where residue two is of a size and charge that the conformationof the peptide mimics the conformation of the peptide in the nativemolecule;

[0115] where residue three is leu or an analog thereof which allowsbinding to antibody number 82D259;

[0116] where X is a linker and where preferably different linkers orlinker method are used at each immunization or where Y is varied betweeneach immunization; and

[0117] where R is a carrier that allows a vigorous immune response.

[0118] This immunogen was used to prime to the peptide and the animalsubsequently boosted with immunogens where linkages are different.

[0119] Molecular Modeling

[0120] Molecular modeling of the peptide was accomplished using theprogram, Hyperchem (Hypercube, Inc. Waterloo, Ontario, Canada). As canbe seen in FIG. 7A, in the native sequence of the glycatedVal-His-Leu-Thr-Pro-Glu-Glu, the Histidine residue is prominent. WhenHistidine is replaced by Phenylalanine, FIG. 7B the residue is stillprominent and not in the interior position one would imagine if thecharge were neutralized by ionic interaction with an interior residue.The threonine at residue four was replaced with a proline to positionthe His imidazole ring to the interior of the folded peptide. FIG. 8Ashows that this placed the imidazole ring of histidine in an interiorposition while prominently displaying the glycated valine and backboneof histidine. One could not model with 2-amino-3-furylproprionic acid(Her) but utilizing phenylalanine as a Her mimic, FIG. 8B the modelindicated that one would achieve the proper orientation and stericconformation (the phenyalanine ring is bulkier than a furan ring and soone would expect that with the furan ring the ring would assume a moreinterior position. Accordingly for an immunogen we utilized the sequenceFructose-Val-2-amino-3-flurylpropionyl-Leu-Pro-Pro-Glu-Glu-Tyr-Tyr-Cys.

[0121] It was maintained that Her, whether 2-amino-3furanylproprionicacid or phenylalanine or some other analog of these, is not an obviousanalog of Histidine in this peptide. 2-amino-3furanylproprionic acid isnot a naturally occurring amino acid and is not commercially available.Her's substitution in the peptide abolished binding with the Milesantibody. Her serves a different function than an analog. An analog isby definition something that is similar in function but not in originand structure in immunochemistry it refers to an equivalent immunologicsubstitute. The purpose of Her is to abolish the immunodominance andcharge of histidine so it does not serve this purpose.

[0122] The first immunization effort with the Her Peptide immunogengenerated only a modest number of viable reactive wells for each fusionand out of 10 animals and fusions only one clone (clone 87) was obtainedthat met the desired specifications. This clone was not stable. Theimmunogenicity (titers) of this immunogen was relatively low and thedesired antibody clones generated by this immunogen were a smallpercentage of the total antibody response. Thus, while this strategymight eventually generate an antibody it was looking for a needle in ahaystack. To increase the chance of success a strategy was developedthat increases the immunogenicity of weak immunogens. This change inimmunizing strategy is thus designed to increase the size of the“haystack” and assumes that the number of “needles” remains at a fixedpercentage of the “hay”. With this strategy one was able to increase thenumber of reactive clones from one fusion to six hundred and to selectsix clones with potential to have high titer, high specificity anddesired reactivity.

[0123] One reason that this strategy may have so successfully assistedin the generation of antibodies to HbA1c is that the weaker theimmunogen, the more important it is that the immune response of theanimal is directed away from the linker. Since the conformation of thepeptide is dependent on the linker and the protein linkage, anotherfunction served by changing the portion of the peptide that serves tolink the peptide to the carrier protein is that changing linkers avoidsboosting to a linker induced epitope conformation.

[0124] The strategy utilized to overcome the weak immunogenicity ofglycated residue peptide has been described in U.S. S. No. 60/059,377Entitled “Method to Improve Immunogenicity of Antigens and AntibodyResponses”. This strategy is hereafter referred to as “focusedimmunization”. In immunizing with poor immunogens the predominantimmunological response may be to the linker or to other non-relevantportions of the peptide immunogen and that one could change the focus bychanging the linker (but not the carrier protein) with eachimmunization. The linker specific response and/or the response to alinker induced epitope conformation is reduced and focused the secondaryresponse on the desired epitope.

[0125] It was not until one immunized utilizing the Focused Immunizationprotocol that one obtained sufficient clones (about 600 reactive clonesfrom fusion number 82D) to allow isolation of a monoclonal 82D259 withthe desired characteristics, i.e., an antibody with increasedspecificity; an antibody which eliminates the need for pretreatment andwhich can be used in a variety of formats for clinical and home testing;and an antibody that can be produced economically.

[0126] Preparation of Immunogens

[0127] Furylacrylic acid, (S)-4-phenylmethyl-2-oxazolidone, pivaloylchloride, FMOC-Cl, 2% Pd—SrCO₃, Raney-Nickel, n-BuLi, KHMDS, triethylamine, 30% H₂O₂, NaOH, HCl, Na₂SO₄, THF, NaHCO₃, acetic acid, Na₂SO₃,trifluroacetic acid, citric acid, and pyridine were purchased fromAldrich. Glucose and â-mercaptoethanol were purchased from Sigma.KLH-maleimide was purchased from Pierce. Ethanol, CH₂Cl₂, ethylacetate,diethyl ether, methanol, and petroleum ether were purchased from VWRScientific. Trisyl azide was prepared as in the literature (Harmon, R.E.; Wellman, G.; Gupta, S. K. J. Org. Chem. 1973, 38, 11-16.) Thepeptide was synthesized by AnaSpec, Inc., 2020 Lundy Avenue, San Jose,Calif. 95131.

Preparation of 3-furylpropionic Acid

[0128] Furylacrylic acid (1 g) was dissolved in 15% NaOH (100 mL, smallamount of ethanol was added to facilitate it dissolve). Hydrogenationwas then carried out with catalytic amount of 2% Pd—SrCO₃ for two days.The mixture was filtered, acidified with 6 N HCl, extracted with CH₂Cl₂,dried over Na₂SO₄, and the solvent was removed. The residue wasrecrystalized from petroleum ether at −20 C as a white powder.

Preparation of(S)-3-(1′-Oxo-3-furylpropyl)-4-phenylmethyl-2-oxazolidinone

[0129] To a solution of 3-furylpropionic acid (163 mg) in THF (4 mL)cooled to −78 C was added triethyl amine (0.21 mL) and pivaloyl chloride(0.16 mL). The mixture was allowed to warmed to 0 C after 15 min. andwas stirred at 0 C for 45 min, then the mixture was cooled back to 78 C.

[0130] To a solution of (S)-4-phenylmethyl-2-oxazolidone (248 mg) in 10mL THF, cooled to −78 C, was added to 0.87 mL n-BuLi (1.6 M in hexane).The resulting solution was added to the above mixture and was stirredfor 10 min. at −78 C. The mixture was then allowed to warm to 0 C andwas stirred for 2 hr. The mixture was quenched with saturated NaHCO₃ anddiluted with diethyl ether. The aqueous layer was extracted with diethylether and the combined organic layer was washed with brine, dried overNa₂SO₄, and concentrated in vacuum. The residue was purified by flashchromatography (1:1 diethyl ether and petroleum ether).

Preparation of [S-(R*,R*)]-3-(2′-azido-1′-oxo-3-furylpropyl)-4phenylmethyl-2-oxazolidinone

[0131] To a solution of(S)-3-(1′oxo-3-furylpropyl)-4-phenylmethyl-2-oxazolidinone (5.7 g) in100 mL THF cooled to −78 C was added to 40 mL of KHMDS (0.5 M intoluene) solution dropwise. The mixture was stirred at −78 C for 20 min.A cold solution of trisyl azide (7.4 g) in 40 mL THF was added to thereaction mixture and was stirred at −78 for 15 min. The reaction mixturewas then quenched with 3.2 mL acetic acid and was allowed to warm up toroom temperature. After stirring for 2 hr., the reaction mixture wasdiluted with diethyl ether, washed with saturated NaHCO₃ brine, driedover Na₂SO₄, and concentrated in vacuum. The residue was purified byflash chromatography (1:1 diethyl ether and petroleum ether) to give theproduct as an oil.

Preparation of (S)-2-azido-3-furylpropionic Acid

[0132] To a solution of[S-(R*,R*)]-3-(2′-azido-1′-oxo-3-furylpropyl)-4-phenylmethyl-2-oxazolidinone(4.6 g) in 150 mL THF and 20 mL H₂O was added 5.5 mL of 30% H₂O₂ at 0 Cand the mixture was stirred for 2 hr. at 0 C. Na₂SO₃ (7.6 g) was addedand then the mixture was stirred for another 10 min. THF was removed,diluted with H₂O, extracted with CH₂Cl₂, acidified with citric acid topH 3, extracted again with ethylacetate, washed with brine, dried overNa₂SO₄, and concentrated in vacuum. The residue was purified by flashchromatography (1% acetic acid in ethylacetate) to give the product asan oil.

Preparation of (S)-2-amino-3-furylpropinonic Acid Trifluroacetic AcidSalt

[0133] To a solution of (S)-2-azido-3-furylpropinonic acid (1.4 g) in200 mL ethanol was added Raney Nickel (0.5 g, washed with H₂O twice andethanol twice). The mixture was hydrogenated overnight. Ethanol wasremoved and the residue was dissolved in H₂O with a small amount oftrifluroacetic acid. The solution was extracted with ethylacetate, thewater was removed, the product was azeotrophed with toluene, and theresidue was dried under vacuum. The product was dissolved in methanoland was titrated with Et₂O to give a white powder.

Preparation of N-FMOC-(S)-2-amino-3-furylpropinonic Acid

[0134] (S)-2-amino-3-furylpropinonic acid trifluroacetic acid salt (0.47g) was dissolved in 30 mL of 10% Na₂CO₃ and 5 mL of dioxane and wascooled to 0 C. A solution of FMOC-Cl (0.53 g) in 10 mL was addeddropwise. The mixture was then stirred for 15 min. at 0 C and 2 hr atroom temperature. The mixture was diluted with H₂O, extracted withdiethyl ether, and acidified with citric acid. The mixture was filteredand washed with H₂O until neutral. The solid was then dissolved inethylacetate, dried over Na₂SO₄, concentrated in vacuo to give theproduct as a white foam.

Preparation of Val-Her-Leu-Pro-Pro-Glu-Glu-Tyr-Tyr-Cys

[0135] The synthesis of this peptide was completed by Ana Spec, Inc.using peptide synthesizer. Preparation of GlycatedVal-Her-Leu-Pro-Pro-Glu-Glu-Tyr-Tyr-Cys PeptideVal-Her-Leu-Pro-Pro-Glu-Glu-Tyr-Tyr-Cys (20 mg) and glucose (86 mg) wasazeotrophed with pyridine twice. The residue was then dissolved inpyridine (2 mL) and the mixture was stirred at dark for 4 days. Pyridinewas then removed.

Preparation of Glycated-Val-Her-Leu-Pro-Pro-Glu-Glu-Tyr-Tyr-Cys and KLHConjugates

[0136] Conjugate 1: 2.5 mg of glycated-peptide in 1 mL of phosphatebuffer (75 mM phosphate, 0.9 M NaCl, pH 7.2) was mixed with 5 mgKLH-maleimide in 0.5 mL H₂O. The mixture was stirred at room temperaturefor 2 hr. To this was then added 1 μL of â-mercaptoethanol (14.3 M) andwas stirred for another 2 hr. The mixture was dialyzed against 4 changesof phosphate buffer (83 mM phosphate, 0.9 M NaCl, pH 7.2).

[0137] Conjugate 2: 4 mg of glycated-peptide in 1 mL of H₂O was mixedwith 4 mg of KLH in 0.4 mL H₂O. To above mixture, 30 mg of EDC was addedand mixture was incubated 4 hr at room temperature with pH kept at5.5-6.0. The mixture was dialyzed against 4 changes of PBS.

Immunization of Animals and Screening for Specific Antibody ImmunizationProtocol

[0138] Mice, Swiss Webster, were injected first withGlycated-Val-Her-Leu-Pro-Pro-Glu-Glu-Tyr-Tyr-Cys-KLH (conjugate #1) at10 microgram per mouse with CFA. After three weeks, mice were boostedwith conjugate #2 at 10 microgram per animal with IFA. Two weeks afterfirst boost mice were bled and antisera were tested in Elisa forantibody titer. Second and third boost were done in three weeksintervals with conjugate #1 at 10 microgram of immunogen per dose.Testing was done two weeks after each boost.

Testing of Mice Bleeds

[0139] Mice sera at different dilutions were incubated with microtiterplates coated with solid phase antigen which was an equal mixture ofGlycated-Val-Her-Leu-Pro-Pro-Glu-Glu-Tyr-Tyr-Cys andGlycated-Val-Phe-Leu-Pro-Pro-Glu-Tyr-Tyr-Cys conjugated to maleimideactivated rabbit gamma globulin. Antibody titers after first boost werein the range of 1:10,000 to 1:100,000 and after second boost were in therange of 1:100,000 to 1,000,000. Mouse #3.4 showed titer 1:1,000,000 andwas used for fusion after third boost.

[0140] The fusion yielded hybridomas that bound both Hb Ao and Hb A1c,as shown by FIG. 9. Hybridoma 82D259 distinguished between HbAo andHbA1c best.

1 8 1 4 PRT Homo sapiens 1 Val His Leu Thr 1 2 15 PRT Homo sapiens 2 ValHis Leu Thr Pro Glu Glu Lys Ser Ala Val Thr Ala Leu Trp 1 5 10 15 3 11PRT Homo sapiens 3 Val His Leu Thr Pro Glu Glu Lys Tyr Tyr Cys 1 5 10 410 PRT Homo sapiens MISC_FEATURE (2)..(2) 2-amino-3-furanylpropionicacid 4 Val Xaa Leu Pro Pro Glu Glu Tyr Tyr Cys 1 5 10 5 10 PRT Homosapiens 5 Val His Leu Thr Pro Glu Glu Tyr Tyr Cys 1 5 10 6 10 PRT Homosapiens 6 Val Phe Leu Thr Pro Glu Glu Tyr Tyr Cys 1 5 10 7 10 PRT Homosapiens 7 Val His Leu Pro Pro Glu Glu Tyr Tyr Cys 1 5 10 8 10 PRT Homosapiens 8 Val Phe Leu Pro Pro Glu Glu Tyr Tyr Cys 1 5 10

We claim:
 1. A method of immunizing to a hapten antigen comprisingimmunizing an animal with the hapten linked to a carrier protein andboosting the immunization with the hapten conjugated to the carrier by adifferent linker or with a different linkage.
 2. The method of claim 1wherein the boosting is done with the hapten conjugated with a differentlinkage.
 3. The method of claim 1 wherein the boosting immunization isdone with a peptide mimic of the hapten.
 4. The method of claim 1wherein the boosting immunization is done with a different linker. 5.The method of claim 1 wherein the initial immunization is done withhapten linked to a carrier protein by a first linkage or linker, asecond immunization is done with hapten linked to the carrier protein bya second linkage or linker, a third immunization is done with haptenlinked to the carrier protein by the first linkage or linker, and afourther immunization is done with hapten linked to the carrier proteinby the second linkage or linker.
 6. The method of claim 1 wherein thehapten is cotinine or a cotinine derivative.
 7. The method of claim 1for producing an antibody to a glycated protein by utilizing animmunogen composed of a glycated peptide mimic of the glycated peptidesequence which is the target epitope, wherein the peptide mimic isconstructed to conformationally mimic the conformation of the peptide inthe native protein, wherein the peptide mimic contains no charged groupsor other immunodominant group, and wherein the peptide mimic isconnected to a spacer sequence equivalent to a peptide spacer of betweenone and thirty amino acids in length, which serves to position thepeptide epitope in a conformation that approximates its conformation inthe native protein.
 8. The method of claim 7 wherein the peptide mimicand spacer are linked to a carrier molecule.
 9. The method of claim 7for producing an antibody to the glycated protein HbA1c, wherein thepeptide mimic comprises amino acid residues sequentially numberedbeginning with one, where residue one is a valine modified by additionof a glucose molecule, where residue two is an analog of Histidine doesnot bear a charge in the immunizing structure, allows orientation of thepeptide so that the immune response can be directed to the side of thepeptide chain oriented oppositely to the ring, and is of a size that theconformation of the peptide mimics the conformation of the peptide inthe native molecule, where residue three is leu or an analog thereofwhich allows binding to an antibody preferentially recognizing Hb A1c,and where residue four is Thr or an analog thereof which allows bindingto antibody number 82D259.
 10. The method of claim 9 wherein the peptidemimic further comprises a peptide spacer of between one and thirty aminoacids in length, which serves to position the peptide epitope in aconformation that approximates its conformation in the native protein.11. The method of claim 10 wherein the peptide mimic is linked to acarrier protein.
 12. The method of claim 9 wherein the histidine analogis 2-amino-3-flurylpropionyl.
 13. A method of immunizing an animal toHbA1c comprising administering to the animal an immunogenic amount ofthe peptide Fructosyl-Val-2-amino-3-furanylproprionicacid-Leu-Pro-Pro-Glu-Glu-Tyr-Tyr-Cys.
 14. The method of claim 1 forimmunizing to a hapten wherein the linkage is selected to provide littleor no antigenic competition.
 15. The method of claim 1 for immunizing toa hapten in which the linker portion of the peptide is selected toprovide the epitope portion of the molecule in the configuration that itappears on the surface of the molecule.
 16. A method of immunizing to ahapten linked to a carrier protein, comprising selecting the linkage tothe carrier protein to provide minimal antigenic competition for immuneresponse and to maintain the epitope portion of the molecule in theconfiguration that it appears in the assay system.
 17. Thehapten-carrier protein conjugates for use in any of the methods ofclaims 1-16.